CN112813068A - Soybean root tissue specific promoter Glyma12g02240 and application thereof - Google Patents

Abstract

The invention discloses a soybean root tissue specific promoter Glyma12g02240 and application thereof, wherein a nucleotide sequence of the promoter Glyma12g02240 is shown as SEQ ID NO. 1. The soybean root tissue specific promoter Glyma12g02240 can enable soybean root tissue to specifically express a GmCaM4 gene, so that the resistance of transgenic soybeans to salt stress is improved.

Description

Soybean root tissue specific promoter Glyma12g02240 and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, and particularly relates to a soybean root tissue specific promoter Glyma12g02240 and application thereof.

Background

Promoters play a very important role in the initiation and regulation of gene transcription, and are important components in transgenic engineering. A promoter is a region of a DNA strand upstream of the coding region of a gene that contains specific sequences that are recognized by transcription factors. The promoter realizes the initiation of transcription and the regulation of a transcription process through the interaction with RNA polymerase and transcription factors, and precisely controls the time, space and intensity of gene expression. The specific nucleotide structure of the promoter region facilitates its binding to RNA polymerase, and the different structures affect its affinity for RNA polymerase binding and thus the level of gene expression.

For many years, a number of promoters have been isolated from a variety of tissues and applied to plant genetic engineering. Promoters affect the transcription process both qualitatively and quantitatively, and the success of transgenic technology depends on their efficient selection and use, both in the field of basic research, in crop improvement and in biopharmaceutical aspects. The promoter has many transcriptional regulatory elements that bind RNA polymerase II at its initiation site. They are important in controlling the expression pattern of genes, activating a gene at the appropriate time and location.

Tissue-specific promoters are generally involved in the regulation of plant growth and development, under the control of which transcription of a gene occurs selectively in a particular tissue or organ. The specific expression mode makes up the defect that the constitutive promoter drives the continuous expression of the exogenous gene to cause adverse effect on plants, and solves the problem of transgenic environmental safety to a certain extent. Tissue-specific promoters generally contain cis-acting elements that control specific expression, the type and location of these elements determining the tissue specificity of promoter expression.

Hormonal or abiotic stress may regulate tissue-specific promoters. For example, the rice MT promoter is wound-induced to be preferentially expressed in roots and flowers. The root-specific promoter PsPR10 enables the expression level of GUS gene to be higher in response to abiotic stress NaCl, PEG6000 and mannitol and responses to SA, ABA and JA.

The soybean genetic transformation in the invention utilizes an agrobacterium-mediated soybean cotyledonary node transformation system, and the soybean GmCaM4 gene is verified to play a positive regulation role in soybean salt stress resistance (Rao S, El-Habbak M H, Havens WM, et al. overexpression of r, GmCaM4 r, in soybean engineering resistance to pathogens and tolerance to salt [ J ]. molecular plant Pathology,2014,15(2):145 and 160.). The resistance of the GmCaM4 gene to salt stress and qRT-PCR are utilized to verify the tissue expression specificity of the soybean root tissue specific promoter and the application of the soybean root tissue specific promoter to the salt stress.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiment of the invention provides a soybean root tissue specific promoter Glyma12g02240 and application thereof. The technical scheme is as follows:

in a first aspect, a soybean root tissue specific promoter Glyma12g02240 is provided, and the nucleotide sequence of the promoter Glyma12g02240 is shown as SEQ ID No. 1.

In a second aspect, there is provided an expression vector comprising the promoter Glyma12g02240 of the first aspect.

Furthermore, the expression vector is a plant expression vector which is pCAMBIA3301-Glyma12g 02240-GUS.

In a third aspect, there is provided a host comprising the expression vector of the second aspect, wherein the host is Agrobacterium tumefaciens GV 3101.

In a fourth aspect, the application of the promoter Glyma12g02240 of the first aspect in preparing transgenic soybean is provided.

In a fifth aspect, there is provided a use of the expression vector of the second aspect in the preparation of transgenic soybean.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the gene with high expression level in soybean roots and low expression level in other tissues is searched through a soybean database soybase (https:// soybase. org/sbt /), and a promoter sequence of 1809bp upstream of an initiation codon ATG of a soybean root tissue specific promoter Glyma12g02240 is cloned. Constructed to a binary expression vector pCAMBIA3301 with GUS gene by enzyme digestion connection. The promoter function verification is carried out in model plants of arabidopsis thaliana and soybean root by an inflorescence infection method of arabidopsis thaliana and a transformation experiment of soybean rhizogenes agrobacterium induced rooting. The soybean root tissue specific promoter Glyma12g02240 can enable soybean root tissue to specifically express a GmCaM4 gene, so that the resistance of transgenic soybeans to salt stress is improved.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below.

FIG. 1 is a vector of the Glyma12g02240 promoter linked to PMD18-T in an example of the present invention;

FIG. 2 is the original map of pCAMBIA3301 vector in the present example;

FIG. 3 shows the results of RT-pcr in the present invention to verify the expression of Glyma12g02240 gene in different tissues;

FIG. 4 is a binary expression vector for transforming Arabidopsis and soybean plants in an example of the present invention;

FIG. 5 shows GUS staining of transgenic Arabidopsis thaliana at T3 generation in the examples of the present invention;

FIG. 6 shows GUS expression in Agrobacterium rhizogenes in examples of the present invention;

FIG. 7 shows the gene expression of GmCaM4 gene in different tissue parts of transgenic soybean plants in the examples of the present invention;

FIG. 8 shows the salt stress resistance of transgenic soybean when GmCaM4 gene is promoted by root specific promoter in the embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Examples

The results of statistics of the expression levels of the Glyma12g02240 gene in different tissues of soybean based on the soybean soybase database are shown in Table 1, and it is assumed that the expression level of the Glyma12g02240 gene in roots is significantly higher than that of other tissue parts as shown in Table 1, and thus the gene is likely to be specifically expressed in roots.

TABLE 1 expression results of soybean gene Glyma12g02240 in different tissues

(1) Gene sequence analysis of specific promoter Glyma12g02240 of soybean root

Primers required for cloning promoter Glyma12g02240 (Pst I cleavage site and BamH I cleavage site and protective base are respectively introduced at 5' ends of forward primer and reverse primer) were designed as follows:

F：5’-AAAACTGCAGCTCTCTCGTCCAAACAGAACATAGA-3’

R：5’-CGCGGATCCTATATAGAGCCCTGTCTTTGACCAA-3’

wherein ACTGCAG and GGATCC are the cleavage sites for Pst I and BamH I, respectively, and the first three bases are the protecting bases.

RNA of the roots of soybean Willimas82 variety was extracted as a cloning template, PCR cloning was performed using synthetic cloning primers (amplification system is shown in Table 2, amplification program is shown in Table 3), PCR product was detected by 1% agarose electrophoresis and then gel recovery was performed, and the product was ligated with pMD18-T for monoclonal test validation.

TABLE 2 amplification System

TABLE 3 amplification procedure

The result shows that the sequence of the promoter Glyma12g02240 is shown as SEQ ID NO. 1.

Promoter analysis software plantaCARE (http:// bioinformatics. psb. agent. be/Webtools/planta/html /), ScanWM-P (Softberry, http:// www/Softberry. com/berry. phtml) was used to predict the presence of cis-elements in the promoter, as predicted in Table 4.

TABLE 4 Soybean Gene Glyma12g02240 promoter cis-element prediction

Name of element	Core sequence	Number of occurrences	Biological function
				RHE	KCACGW
	1	Root hair distribution related elements
			M1-motif	TACATGT		1	Cis-regulatory regions associated with root-specific expression
M2-motif	ACTTATA							1	Cis-regulatory regions associated with root-specific expression
			M3-motif	ATAAAAC	0	Cis-regulatory regions associated with root-specific expression
telo-box	AAACCCTAA	0					Root primordial expression correlation

(2) Analysis of expression of soybean root-specific promoter Glyma12g02240 in Arabidopsis thaliana

2.1 the promoter sequence was ligated to the expression vector pCAMBIA3301-Glyma12g02240-GUS by enzymatic ligation and transformed into Agrobacterium tumefaciens GV 3101.

2.1.1 expression vector pCAMBIA3301-Glyma12g02240-GUS construction procedure

2.1.1.1 Glyma12g02240 promoter ligated into pMD18-T vector

The genomic DNA of soybean variety WS82 was used as a template, and the amplification system (Table 5) and PCR program (Table 6) were as follows:

TABLE 5 amplification System

TABLE 6PCR amplification procedure

50 μ L of PCR product was detected and recovered on 1.5% agarose gel electrophoresis, and the promoter fragment obtained by gel cutting recovery was ligated into pMD18-T vector, the map of which is shown in FIG. 1, in the system shown in Table 7.

Table 7 target promoter connection system for connecting PMD18-T vector

After the ligation system was added to the PCR tube, it was centrifuged instantaneously and placed in a PCR apparatus for ligation overnight at 16 ℃.

2.1.1.2 the pCAMBIA3301 carrier is modified into pCAMBIA3301-GUS

The pCAMBIA3301 vector original map is shown in FIG. 2. According to the vector, a CaMV 35s promoter is used for starting GUS gene expression, and when a promoter GUS expression vector is constructed, an appropriate enzyme cutting site is selected to replace the CaMV 35s promoter with a candidate tissue specific promoter, so that the condition that the candidate promoter starts GUS gene expression is tested. However, in view of enzyme cutting site analysis of the sequence of the candidate promoter in the experiment, enzyme cutting sites such as Hind III, EcoR I, Nco I and the like are found in the promoter sequence, and if the enzyme cutting sites are used, the promoter sequence is incomplete, so that the pCAMBIA3301 vector is modified in the experiment, and enzyme cutting sites which are not in the candidate promoter sequence are selected: PstI and BamHI, used to insert candidate promoters.

Carrying out double enzyme digestion on the vector by using Xma I and Nco I, removing a 35s promoter and enzyme digestion sites around the promoter, designing a primer, cloning to obtain a sequence of 'Xma I + Pst I + CaMV 35s promoter + BamH I + Nco I', carrying out double enzyme digestion on the sequence and a vector framework, and reconnecting to the vector to complete the transformation. The primer sequences (Table 8), cleavage and ligation system (Table 9) were as follows:

TABLE 8 vector engineering primer sequences

TABLE 9 double enzyme digestion System

The digested vector was detected on 1.5% agarose gel electrophoresis, photographed under an ultraviolet gel imager, cut under an ultraviolet lamp to obtain a larger vector backbone (about 10000bp), recovered with a kit (reference 2.2.4), and the double digested DNA fragment and pCAMBIA3301 vector fragment were ligated at a molar ratio of 3:1, as shown in Table 10.

Ligation System of fragments of the order of Table 10 with vector fragments

And transforming the connected vector into DH5 alpha escherichia coli competent cells (refer to 2.2.5.2), carrying out sequencing verification, carrying out small-scale plasmid extraction, and storing at-20 ℃ to complete vector transformation.

Construction of pCAMBIA3301-Glyma12g02240-GUS vector

The pMD18-T vector with the target promoter and the modified pCAMBIA3301 vector are subjected to double enzyme digestion by using Pst I and BamH I, and the enzyme digestion system is shown in Table 11.

TABLE 11 double enzyme digestion System

The enzyme digestion system was detected on 1.5% agarose gel electrophoresis and photographed under an ultraviolet gel imager, the larger vector backbone (approximately 10000bp) and the candidate promoter fragment (approximately 2000bp, slightly smaller than the T vector backbone) excised from the pMD18-T vector were excised under an ultraviolet lamp and recovered with a kit (reference 2.2.4), and the double enzyme digested promoter fragment and the pCAMBIA3301 vector fragment were ligated in a molar ratio of 3:1, the ligation system is as in table 12.

TABLE 12 ligation of promoter fragments with vector fragments

And transforming the connected vector into escherichia coli DH5 alpha competent cells (refer to 2.2.5.2), selecting a single clone, detecting bacterial liquid by using a promoter specific primer, carrying out sequencing verification, carrying out small-extraction on the plasmid, storing the plasmid at-20 ℃, and completing construction of an expression vector pCAMBIA3301-Glyma12g 02240-GUS.

Construction of 2.1.1.4 pCAMBIA3301-Glyma12g02240-CaM4-GUS vector

Cloning primers using GmCaM4 were cloned into pMD18-T vector as above, with primers as in Table 13.

TABLE 13 primer sequences

Forward primer (F-BamHII)	cgggatcccgATGGCAGATATCCTGAGTGAAGAAC
		Reverse primer (R-NcoI)	catgccatggcatgTCGAACGGTCATCATCATCTTG

The cloned pMD18-T and pCAMBIA3301-Glyma12g02240-GUS were digested simultaneously with BamHI and Nco I, and the fragments were recovered and ligated as described above. The vector was verified for correctness by PCR and sequencing.

2.2 transforming Arabidopsis thaliana Col plant by Agrobacterium mediation method, the method is as follows:

1. the day before transformation, the wild type Col Arabidopsis seedlings to be used were watered with a sufficient amount of water.

2. About 10 wild arabidopsis thaliana plants are transformed, enough inflorescences are selected to form the wild arabidopsis thaliana at the initial flowering stage, and the grown fruit pods and open flowers of the plants to be infected are cut off.

3. Pouring the infection liquid with the agrobacterium tumefaciens GV3101 thalli into a small beaker, soaking the overground part of the arabidopsis into the infection liquid, infecting for 3min, taking out, immediately wrapping the plant with a preservative film, keeping flat and shading, uncovering the preservative film after 18h (preferably not more than 20h to prevent the plant from wilting), and continuously culturing under normal conditions.

Then 2 per mill of Basta solution (herbicide) is sprayed on plants, and transgenic positive plants with pCAMBIA3301-Glyma12g02240-GUS vectors can express herbicide-resistant genes (bar genes) and grow normally; plants with failed transgenosis will wilt and die until T3 homozygous lines are screened.

GUS staining is to perform GUS histochemical staining on plants by using a GUS staining kit, and 1mL of GUS staining concentrated solution (50X) is added into 50mL of GUS staining buffer solution (namely diluted by 50 times), and the mixture is uniformly mixed to prepare a GUS staining solution. Taking out the plant tissue material, cleaning, especially taking care of cleaning the plant root system, slowly washing soil with water, and taking care of gentle operation to avoid damaging the plant root system. Placing the cleaned plant tissue material into a culture dish, adding 90% acetone, covering the plant tissue material with the liquid surface, and standing at 4 deg.C for 20min to fix the material and prevent GUS signal diffusion. The acetone was decanted, washed off with GUS rinse and repeated twice. Pouring the cleaning solution in the culture dish, adding the GUS staining solution to enable the GUS staining solution to submerge plant tissue materials, wrapping the culture dish by tinfoil, putting the culture dish into a 37 ℃ incubator, frequently observing the staining condition of the plant tissue materials, and if the plant tissue materials are stained, decolorizing can be carried out, and the plant tissue materials can be stained overnight. Pouring out GUS staining solution, sucking residual GUS staining solution with a pipette, adding 70% ethanol, and decolorizing, wherein the ethanol is replaced at first, and the time for decolorizing is at least 3-5 times until complete decolorizing (negative control material is white). After completion of decolorization, photographs were taken with a microscope.

(3) Analysis of expression of soybean root-specific promoter Glyma12g02240 in soybean root

Firstly, RNA of different tissues of soybean is extracted to be used as an RT-pcr template, the expression condition of Glyma12g02240 gene is semi-quantitatively detected, ACT11 is used as an internal reference, and the primers are as follows:

Glyma12g02240-F：5’-TTGTTGAGGGCTGTGATGG-3’

Glyma12g02240-R：5’-GAATTTGATATGTCGGGTACTGA-3’

ACT11-F：5’-ATCTTGACTGAGCGTGGTTATTCC-3’

ACT11-R：5’-GCTGGTCCTGGCTGTCTCC-3’

the PCR products were verified by running 1% agarose gel electrophoresis, and the results are shown in FIG. 3.

(4) The agrobacterium rhizogenes-mediated soybean genetic transformation method is as follows (the formula of the culture medium is shown in table 14):

1. a proper amount of plump and yellow-moist soybean Willimas82 seeds without spots and cracks were selected.

2. Putting the selected seeds into a culture dish (the soybeans are not too much and only need to be spread in a layer, otherwise, the soybeans are not completely sterilized when the soybeans are stacked together), opening a culture dish cover, and putting the culture dish cover into a sealed tank; 40mL of sodium hypochlorite solution (NaClO) was placed in a 50mL centrifuge tube and also in a sealed pot, and 1.6mL of concentrated hydrochloric acid was added dropwise to the sodium hypochlorite solution to generate chlorine gas for sterilization (2HCl + NaClO ═ Cl)₂↑+NaCl+H₂O), sealing the can opening with preservative film, sealing for 18-20h, and taking care to operate in a fume hood. GM medium was prepared the day, plates were poured overnight, and forceps were sterilized for use.

3. Opening the sealed tank, quickly covering the culture dish cover, taking out the culture dish, placing the culture dish into a sterile operating platform, blowing for 30min, removing the smell of chlorine, burning and sterilizing the sterilized tweezers on the outer flame of an alcohol lamp again, after the tweezers are cooled, clamping the seeds, placing the hilum downwards, planting the seeds in a GM culture medium, and paying attention to the fact that the seeds are slightly inserted into the culture medium, wherein 10 seeds are planted in each culture dish.

4. Placing the culture dish with the planted seeds in a paper box, culturing in dark at 25 ℃ for five days, checking the state of the seeds once a day during the dark culture period, and taking out the polluted seeds in time.

5. Tweezers, a knife, a blue tip, cut filter paper (placed in a petri dish), a proper amount of petri dish and a 50mL centrifuge tube are prepared, and sterilized for standby at 121 ℃ for 20 min.

6. Pouring the LCCM culture medium into a sterile 50mL centrifuge tube (one centrifuge tube is needed for each plasmid transformed agrobacterium), sucking 2-4mL of the LCCM culture medium onto a plate coated with bacterial liquid by using a gun head, blowing and beating the bacteria uniformly, and putting the bacteria into the 50mL centrifuge tube for marking.

7. Taking the germinated soybean seeds of 5-6d, cutting off the soybean seeds from the hypocotyls at the position of 0.3-0.5 cm (cutting on a cover of a sterilized culture dish, and optionally filling a piece of sterilized filter paper), cutting off the cotyledon in two parts (one seed is divided into two parts), and removing the terminal bud. And lightly scratching 5-7 wounds at cotyledon nodes by using a blade, and scratching a plurality of wounds on the surfaces of the cotyledon nodes to obtain the explants.

8. Pouring 50mL of the infection liquid LCCM culture medium with bacteria into a sterilized culture dish, placing the cut explants in the culture dish, and soaking for 20-30min, wherein the culture dish needs to be shaken frequently during the period to prevent the bacteria liquid from precipitating to the bottom. The cover of the SCCM culture medium is opened, filter papers are respectively paved on the cover and the SCCM culture medium, the transfected cotyledon node is firstly put on the filter paper of the cover to dry in the shade, then the cotyledon node is inoculated on the SCCM culture medium with a layer of sterile filter paper (10 petals of seeds are put on each culture dish), the culture dish is sealed by a sealing film and is put in a paper box to be cultured in the dark for 5 days at the temperature of 25 ℃.

9. During the preparation, enough liquid and solid hairy root culture medium is prepared, and tweezers, a knife, a blue tip, cut filter paper (placed in a culture dish) and a proper amount of conical flask are sterilized for later use.

10. Clamping beans out by using sterilized tweezers, placing the beans in a conical flask or an open flask, washing off bacteria on the beans by using a rooting medium (liquid, added with Cef and Carben resistance) for three times, shaking and rinsing for the first two times, soaking for half an hour for the third time, frequently shaking the conical flask in the period, clamping the beans after cleaning, placing the beans on sterilized filter paper for airing, cutting off a newly grown hypocotyl (only keeping about 0.5 cm), downwards arranging the hypocotyl of the aired and cut beans, obliquely inserting the bean into a solid rooting medium, and inserting 5 petals of seeds into each rooting medium.

11. Sealing the rooting culture medium, placing the rooting culture medium in an incubator at 25 ℃, illuminating for 16h/d, culturing for 8h/d in the dark, growing hairy roots in about 15-20 days, and then performing GUS histochemical staining observation on the plants by using a GUS staining kit. The results of the experiment are shown in FIG. 5.

TABLE 14 culture medium formula for experiments on soybean infection by Agrobacterium rhizogenes

(5) Agrobacterium-mediated transformation of soybean cotyledonary nodes (the lower medium differs from the upper medium, one for rooting and one for root carcinoma)

The soybean genetic transformation is carried out by adopting an agrobacterium-mediated soybean half-seed method, and a transformation receptor is a susceptible cultivated soybean variety williams 82.

Firstly, the selected smooth and round soybean seeds collected in the current year without black spots are sterilized in a dryer by chlorine gas (100ml of sodium hypochlorite is added with 3.5ml of concentrated HCL for reaction) for 12 to 16 hours, the soybean seeds are opened and aired for 20 to 30min on an aseptic super clean bench, then hilum is planted in GM culture medium (B5 salt, B5 vitamin, 30g/L sucrose, 3.9g/LMES, 5g/L agar powder, pH 5.8) at 23 ℃ for dark culture for 18 to 20 hours. Taking out germinated soybean seeds the next day, slowly cutting the seeds into two symmetrical halves along the hilum part by using a sterilized scalpel, slightly scratching 3-6 knives at the growth point part of cotyledon node by using the scalpel, and then placing the seeds scratched by the knives in agrobacterium with OD being about 08 for infecting for 15-20min at 60 rmp.

② transferring the soybean seeds infected by the agrobacterium liquid to CCM culture medium (B5 vitamin, B5 salt, 30g/L sucrose, 0.25mg/L GA) paved with sterilized filter paper by using clean tweezers₃3.9g/L MES, 1.67mg/L BAP, 154.2mg/L DTT, 400mg/L cysteine, 200. mu. mol/LAS, 5g/L agar powder, pH 5.4) (seed coat removed), and incubated at 24 ℃ in an incubator for 5 days in the dark.

③ transfer the well-grown explants to SIM induction medium (B5 salt, B5 vitamin, 30g/L sucrose, 0.59g/LMES, 1.67mg/LBAP, 250mg/L cefuromycin, 8g/L agar powder, 100mg/L LTimentin, 6mg/L glufosinate, pH 5.7) with paraxial face up, and culture for about 14 days at 25 ℃ under 16/8h light-dark condition.

And fourthly, taking out the explants with good development state (more calluses and green), cutting off redundant hypocotyls at the lower part, and transferring the explants to an SIM induction culture medium again to culture the explants for about 14 days under the condition of continuous normal growth.

Fifthly, transferring the cluster buds generated from the explants in good state to SEM bud elongation culture medium (MS salt, MS vitamins, 50mg/L aspartic acid, 30g/L sucrose, 0.59g/LMES, 0.5mg/L GA)₃50 mg/LL-glutamic acid, 100mg/LTimentin, 0.1mg/LIAA, 1.0mg/L zeatin nucleoside, 250mg/L cefuroxime, 6mg/L phosphinothricin and 8g/L agar powder, and the pH value is 5.7), and the culture conditions are 25 ℃ and 16/8h light-dark cycle culture for 14 days.

When the resistant cluster buds grow to 3-5 cm (and the cluster buds grow well and enough green leaves exist), the cluster buds are quickly cut off from the callus by a sterilized scalpel, soaked in IBA with the concentration of 1mg/L for 30s, transferred to an RM rooting medium (MS salt, 20g/L sucrose, 50mg/L aspartic acid, 0.59g/LMES, 1.0mg/L IBA, 50mg/L L-glutamic acid, 3g/L plant gel, pH 5.6) and continuously cultured for 16/8h in a light-dark period at 25 ℃. When strong white roots (the number of the roots is enough and strong) grow out from the resistant buds, transplanting the strong white roots into a greenhouse for normal condition culture after greenhouse seedling hardening.

The formulation of each of the media used above is shown in Table 15. MS synthetic salt was purchased from Sigma under the trade designation M5524. The B5 synthetic salt was purchased from Sigma under the trade designation G5768.

TABLE 15 culture Medium formulation

(6) Expression condition analysis of soybean root specific promoter Glyma12g02240 in different tissues of transgenic soybean and salt stress resistance analysis thereof

Firstly, RNA of different tissues of transgenic soybeans is extracted, a reverse transcription kit is utilized to transcribe cDNA to be used as a template of qRT-PCR (reaction system shown in table 16) to detect the expression condition of a GmCaM4 gene, GmACT11 is used as an internal reference, and GmCaM4 gene primers are as follows:

F：5’-CACAATAAAGCCACGAGTATGG-3’

R：5’-GCTCAATCTTAGCTGGGCTATA-3’

TABLE 16 real-timePCR reaction systems

Reagent	Dosage of
		SYBR Premix DimerEraser(2×)	5μL
Forward primer (2.5. mu.M)	1μL
		Reverse primer (2.5. mu.M)	1μL
cDNA template	0.3μL
		Water (W)	To 10 μ L

The soybean GmACT11 is used as an internal reference, the reaction conditions adopt a three-step method, and each pair of primers is repeated at least 3 times to obtain a credible experimental result. Basic procedure of the 3-step method: 1 minute at 95 ℃; at 95 deg.C, 5 seconds, 60 deg.C, 10 seconds, 72 deg.C, 30 seconds, 40 cycles; 72 ℃ -melt.

The expression result of the GmCaM4 gene in the transgenic material is shown in figure 7.

The salt stress resistance experiment method of the transgenic material comprises the following steps: after transgenic material and transgenic recipient material Willians82 grew to the second two-to-three double leaf period, watering was stopped to dry the soil, once dry, plants were treated with saline solution (150mM NaCl) once every two days for 10 days, and then watered once a day to observe the plants for re-watering growth, the results are shown in fig. 8.

The gene with high expression level in soybean roots and low expression level in other tissues is searched through a soybean database soybase (https:// soybase. org/sbt /), and a promoter sequence of 1809bp upstream of an initiation codon ATG of a soybean root tissue specific promoter Glyma12g02240 is cloned. Constructed to a binary expression vector pCAMBIA3301 with GUS gene by enzyme digestion connection. The promoter function verification is carried out in model plants of arabidopsis thaliana and soybean root by an inflorescence infection method of arabidopsis thaliana and a transformation experiment of soybean rhizogenes agrobacterium induced rooting. The soybean root tissue specific promoter Glyma12g02240 can enable soybean root tissue to specifically express a GmCaM4 gene, so that the resistance of transgenic soybeans to salt stress is improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> soybean root tissue specific promoter Glyma12g02240 and application thereof

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aaaattaagt aaaatatctt ataaaaatac attttatatc taaaagtatt tttattactc 1620

gaaataaaag tagtaatgtc acaatcaatt tttaaaataa ggttacctat tggagactag 1680

aatcaatgaa tcgaagcata cagtatatgt ttattgttta gtttaagaaa gcaagtttgg 1740

ctttagttgg tggaaaaagg caaaggcaat tcacatttga gtatttggtc aaagacaggg 1800

ctctatata 1809

Claims (6)

1. The soybean root tissue specific promoter Glyma12g02240 is characterized in that the nucleotide sequence of the promoter Glyma12g02240 is shown as SEQ ID NO. 1.

2. An expression vector comprising the promoter Glyma12g02240 according to claim 1.

3. The expression vector of claim 2, wherein the expression vector is a plant expression vector and the plant expression vector is pCAMBIA3301-Glyma12g 02240-GUS.

4. A host comprising the expression vector of any one of claims 2 or 3, wherein the host is Agrobacterium tumefaciens GV 3101.

5. Use of the promoter Glyma12g02240 according to claim 1 for the preparation of transgenic soybean.

6. Use of an expression vector according to any one of claims 2 or 3 for the preparation of transgenic soybean.

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